Pergamon 0306-4522(93)E0018-L

Neuroscience Vol. 59, No. 4, pp. 10.5-1070, 1994 Elsevier Science Ltd

Copyright 0 1994 IBRO Printed in Great Britain. All rights reserved

0306-4522/94 $6.00 + 0.00

BRAIN ACTIVITY PATTERNS IN FLYING,

ECHOLOCATING BATS (PTEROiVOTUS PARNELLII):

ASSESSMENT BY HIGH RESOLUTION

AUTORADIOGRAPHIC IMAGING WITH [ 3H]2-DEOXYGLUCOSE

G. E. DUNCAN*~$ and 0. W. HENSON*

*Department of Cell Biology and Anatomy and TBrain and Development Research Center, University of North Carolina at Chapel Hill, U.S.A.

Abstract-Brain activity patterns during echolocation and flight were assessed in mustached bats (Pterono~usparnelliipurneNii). Bats were injected intraperitoneally with [ ‘H]2-deoxyglucose and restrained in a foam holder or allowed to fly for 20 min. Under resting conditions, low levels of [3H]2-deoxyglucose uptake were observed throughout the forebrain but relatively high uptake was found in brainstem auditory and vestibular centers. In flying, echolocating bats, marked increases in regional [‘H]2-deoxyglucose uptake were apparent. All structures of the classical ascending auditory pathway were intensely labeled in autoradiograms. Other brain regions that exhibited high [‘H]2-deoxyglucose uptake in flying bats included the cingulate cortex, stratum lacunosum-moleculare of the hippocampus, thalamus, cau- date-putamen, superior colliculus, pontine reticular formation, nucleus ambiguus, parts of the midbrain central gray, and cerebellum. In the cerebellum, the most prominent increase in [3H]2-deoxyglucose uptake was found in discrete patches of the granule cell layer.

The results provide the first overview of brain activity patterns during echolocation and flight in bats. In addition, uptake of [ “C]fluorodeoxyglucose was used to compare brain activity patterns in flying bats to bats that were imaging their environment via biosonar while hanging in a wire cage. The echolocat- ing-not-flying bats emitted 6931 k 1226 pulses in 20 min compared to 8972 k 1273 pulses in 20min for flying bats. The uptake of the metabolic marker was significantly more in the flying bats compared to the emitting-not-flying bats in the medial geniculate, superior colliculus, auditory cortex, cingulate cortex and thalamus. In the nucleus ambiguus, cochlear nucleus, and inferior colliculus, uptake was similar for the flying and emitting-not-flying bats. These results suggest that the high metabolic activity observed in forebrain auditory regions of flying bats is related in part to neural processes that involve sensory motor integration during flight and not simply the perception of acoustic information.

Chiroptera are the only mammals specialized for flight and many use biosonar to image their environ- ment and to detect, identify and track prey. Mus- tached bats (Pteronotus parnellii parnellii) have attracted special interest because of their complex ultrasonic biosonar signals and their ability to fly and hunt insects in ‘cluttered’ tropical environments.‘9*24 In addition, mustached bats Doppler-shift compen- sate to keep the returning echoes in sharply tuned portions of their hearing range.26~27~28~34~40.52.56 Echolo- cation and a variety of associated compensations25,3’ are dependent not only on audition, but also on precise vocalization and integration of acoustic and motor systems. The identification of specific vocaliza- tion and acoustic centers in the brain has been classically dependent on neuroanatomical mapping

fTo whom correspondence should be addressed at: Depart- ment of Cell Biology and Anatomy, CB 7090, University of North Carolina, Chapel Hill, NC 27599, U.S.A.

Abbreviations: CF, constant frequency, 2-DC, 2-deoxyglu- case; FDG, fluorodeoxyglucose; FM, frequency modu- lated.

of functional responses with electrophysiological techniques. Such studies require the presentation of artificial stimuli and recording of responses from restrained animals. The activity of the various audi- tory and vocalization centers under echolocative con- ditions during flight is relatively unknown.

One approach for examining topographic patterns

of brain activity in freely behaving animals is the autoradiographic 2-deoxyglucose (2-DG) tech- nique. ‘J’ This technique is based on the fact that the adult brain uses glucose almost exclusively as an energy source and that radiolabeled 2-DG and metabolites accumulate in brain tissue in proportion to the rate of glucose utilization. The 2-DG technique has been used extensively to examine brain activity patterns in response to auditory stimulation in mam- mals (for examples see Refs 3, 6, 21, 43-46). How- ever, limited information exists for brain metabolic patterns after acoustic stimulation in bats. The only 2-DG study available for Pteronotus parnellii exam- ined passive reception of acoustic signals. In these experiments, some brain regions known to be

NSC 59,&J 1051

1052 G. E. DUNCAN and 0. W. HEMON

involved in auditory information processing, such as the auditory cortex and medial geniculate body, did not show signs of activation3’ In contrast, increased 2-DG uptake was observed in the auditory cortex of the horseshoe bat during echolocation.

In the present investigation, brain activity patterns in flying, echolocating mustached bats were examined by autoradiographic imaging of 2-DG uptake. A high resolution technique was used that facilitated analysis of patterns of activation. ‘.‘w This investigation pro- vides an overview of the metabolic activity in brain regions during flight. In addition, brain activity pat- terns were compared in flying-echolocating bats and in bats that were echolocating but not flying.

EXPERIMENTAL PROCEDURES

Mustached bats (P. p. parnellii) were captured in Jamaica and housed in an environmentally controlled room (9O”C, 80% relative humidity) for periods of two to six months before the experiments. Animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of North Carolina Institutional Animal Care &ommjttee. Six bats, weighing 12-14 g were injected i.p. with 200 PCi of [‘H]Z-DG (30 Ci/mmol, American Radiolabeled Chemicals, St Louis, U.S.A.). Three of the bats were gently restrained in a foam holder and the other three bats were allowed to fly in a laboratory room (approximately 200 ft”). The bio- sonar vocalizations were measured for one bat under the restrained condition and 122 pulses were emitted after injection of 2-DG. The other two bats in the resting condition were watched carefully and also rarely exhibited the characteristic mouth movements that occur during emission of echolocation signals. Unrestrained animals flew continuously during the 20-min period except for occasional brief rest periods (lo-30 s). Although biosonar emissions were not measured for the hying bats in part of the investigation, we have found under identical experimental conditions that mustached bats emit 7000-9000 pulses during a 20-min flight period (see below). Under both flying and resting conditions the room lights were on. Each bat was killed by decapitation 20 min after injection of [‘H]2- DG and the brain was removed and frozen on an aluminum block cooled with liquid nitrogen.

Bats were injected i.p. with 85 FCi of [ “Cjfluorodeoxy- elucose (FDG. 300 mCi/mmol, American Radiolabeled Chemicals) and placed in a wire cage (25cm x 25cm x 50 cm, n = 3) restrained in a foam holder (n = 1) or allowed to fly in a laboratory room (n = 3, see above). The kinetic properties of FDG may offer advantages over 2-DG for use in metabolic mapping studies.2.‘u.3z One purpose of our study was to compare qualitatively the ability of two compounds to image functional activity during echolocation and flight in bats. All bats were killed 20 min after the injection of FDG. Bats hanging in the wire cage emitted biosonar pulses spontaneously much of the 20-min period, but when spon- taneous emissions ceased, the bats were induced to keep emitting by waving objects or by gentiy rocking the cage. In these experiments biosonar emissions were recorded on magnetic tape and played back to an electronic counter to determine the number of pulses emitted (General Radio Il92B counter).

High resolution autoradiographlj Coronal sections of brains (10 em) were cut in a cryostat

and thaw-mounted on segments of Kodak SR-5 Industrex

film that were attached to microscope slides with Silicone glue. Details of the autoradiographic procedures have been described previous1y.s.” Sections mounted on lilm were stored in light-tight desiccator boxes before photographic processing. Autoradiograms were developed for IOmin in Kodak D-19. rinsed in water, and fixed for 30 min in Kodak general purpose fixer. For photographic documentation ol autoradiograms, photomicrographs were taken with a Wild M7-S microscope with Kodak Technical Pan film. Prints were made using Kodabrome II RC paper (contrast grade F2) for all conditions, and exposure times were used to reproduce the relative optical densities among the different brain regions in the original autoradiog~~ms.

The amount of radioactivity in autoradiograms was as- sessed by digital image analysis using a system from Imaging Research, Inc., by reference to standards containing known amounts of radioactivity (Amershdm 14C microscate stdn- ddrds). Data are expressed as ratios of radioactivity in gray matter regions to that in the corpus callosum. Statistical comparisons of different treatment conditions were made with repeated ANOVA.

RESUWS

Comparison of‘ activity patterns in frying and resting buts

Cortical regions. Under resting conditions there was little regional variation in 2-DG uptake in corti- cal areas. In general, there were no regions of high 2-DG uptake but a band of slightly higher optical density was seen in autoradiograms in the middle layers of the auditory cortex, compared with other layers and with surrounding regions. In flying bats, marked increases in f3H]2-DG uptake were observed in specific regions of the frontal cortex, anterior cingulate cortex, and olfactory cortex (Table 1; Fig. 1). The extreme anterior part of the medial

Table 1. Relative uptake of 2-deoxyglucose in flying and resting bats

Brain Region Resting Flying

Cingufate i .4t + 0.06 2.06 + 0.11 cortex, ventral

Cingulate I .29 + 0. IO 1.40 + 0.04 cortex, dorsal

Auditory cortex I .63 + 0.06 2.45 + 0.05 Retrosplenial 1.35+0.06 1.89+0.16

cortex Medial 1.44+0.04 2.37 + 0.06

geniculate Inferior 2.21 -to.10 3.33 +0.19

colliculus Superior

coIliculus Ambiguus

nucleus

I .32 + 0.04 2.23 + 0.12

1.41 + 0.04 2.17 + 0.07

Data are expressed as ratios of radioactivity in the brain regions relative to that in the corpus callosum and are mean + S.E.M. with n = 3 for each condition. Repeated-measures ANOVA indicates a significant difference be- tween the flying and resting conditions (P < 0.001).

2-DG uptake in flying bats 1053

Fig. I(A-H). Caption on page 105.5

Fig. 1(1-P). Cupfion opposile.

2-DG uptake in flying bats 1055

frontal cortex showed high optical density, and this density extended posteriorly into the cingulate cortex. Within the cingulate cortex, there were variations in patterns of 2-DG uptake at different rostral-caudal levels (Fig. 1). In the rostra1 cingulate cortex, high 2-DG uptake occurred only in the ventral half (Fig. ID, F). Caudally, at the level of the hippo- campus, high optical density in the cingulate cortex was restricted to a narrow region immediately dorsal to the corpus callosum (Fig. lH, J). The densely labeled auditory cortex contrasted sharply with low optical density in the adjacent cortical tissue medial to the prominent sulcus that defines the dorsal limit of the auditory cortex. A sharply curved boundary of high uptake was also evident in the region ventral to the sulcus (Fig. lH, J).

Caudate-putamen and claustrum. In contrast to the uniform and low 2-DG uptake in the resting condition, high uptake was found in the caudate- putamen of flying bats and a distinctive pattern was observed in autoradiograms (Fig. 1 D, F). The highest uptake in flying bats was found in the dorsal and medial part of the caudate-putamen. High uptake was also observed in the claustrum of flying bats, whereas this region was weakly labeled in auto- radiograms prepared from brains of resting bats (Fig. lE, F).

Thalamus and subthalamic regions. In resting bats, low levels of 2-DG accumulated in the thalamus. In flying bats very high 2-DG uptake occurred in wide- spread thalamic regions (Fig. lJ, L). In the medial geniculate nucleus, discrete patterns were observed; the dorsomedial part was more heavily labeled than the ventrolateral part (Fig. 2). The subthalamic nucleus and zona incerta were heavily labeled in autoradiograms from flying bats (Fig. lN, P).

Hippocampus and amygdata. Under resting con- ditions, low 2-DG uptake occurred in all regions of the hippocampus. In flying bats, high uptake was found in the stratum lacunosum-moleculare of the hippocampus, but relatively low uptake was observed in other hippocampal regions (Fig. I L, N, P). Low

2-DG uptake was found in the amygdala of both resting and flying bats (Fig. lK, L).

Inferior and superior colliculi. The inferior collicu- lus consistently exhibited relatively high 2-DG uptake in bats under resting conditions (Fig. 3). However, in flying bats much greater uptake was observed. (Table 1; Figs 3 and 4). Within the inferior colliculus of flying bats, distinct and consistent patterns of 2-DC uptake were observed at different rostral%audal levels (Fig. 4). The dorsal cortex of the inferior colliculus was weakly labeled relative to the central nucleus (Fig. 4). In the superior colliculus of resting bats, 2-DG uptake was low, except for a thin band on the dorsal surface (Fig. 10). In the flying bats the superior colliculus was one of the most densely labeled regions in autoradiograms (Table 1; Fig. lN, P).

Other brainstem structures. In resting bats, the median raphe, at the level of the inferior colliculus, exhibited relatively high 2-DG uptake; in addition, all vestibular and auditory centers were densely labeled (Fig. 3). Optical densities associated with the superior olivary complex (Fig. 3E, F) and vestibular nuclei (Fig. 3K-M) were similar in the flying and resting bats. In contrast, the pontine reticular formation was weakly labeled in the resting bats and showed greatly increased 2-DG uptake in flying bats (Fig. 3A-D). The nucleus ambiguus (Fig. 31-M) showed markedly increased 2-DG uptake in flying compared to resting bats.

Cerebellum. In resting and flying bats there was relatively high 2-DG uptake in the anterior lobules of the vermis (lobulus centralis and culmen), the parts deeply wedged between the large inferior colliculi (Fig. 3E, F). In addition, the deep cerebellar nuclei showed relatively high 2-DG uptake in resting and in flying bats (Fig. 31-L).

The patterns of 2-DG uptake in the specific regions of the cerebellar cortex were strikingly different in the resting compared to flying bats (Figs 3 and 5). In flying bats, patches of high 2-DG uptake were ob- served in specific regions of the granule cell layer

Abbreviations used in figures A amygdala C auditory cortex AM ambiguus nucleus CING cingulate cortex CP caudateputamen CN cochlear nucleus DC deep cerebellar nuclei FC frontal cortex H hypothalamus HI hippocampus IC inferior colliculus LL lateral lemniscus MG medial geniculate body

MN median raphe PCG pontine central gray PM paramedian fissure of cerebellum PRF pontine reticular formation RS retrosplenial cortex S sulcus SC superior colliculus SLM stratum lacunosum-moleculare of hippocampus ST subthalamic nucleus T thalamus V vermis of cerebellum VN vestibular nucluei ZI zona inserta

Fig. 1. Autoradiograms of coronal sections through the forebrain of resting (left panels) and flying (right panels) bats. Dark regions represent high uptake of [‘H]2-DG. Note the uniformly low 2-DG uptake in

the resting bat and marked increase in uptake of the compound in select regions of the flying bat.

1056 G. E. DUNCAN snd 0. W. HENSON

Fig. 2. Autora~iograms of the medial geniculate nucleus of a flying bat. Sections are progressively caudaf from A to D. Photomicrographs are under-exposed to illustrate patterns of2-DG uptake. Note the dense

labeling in the dorsomedial part and the lighter labeling in the ventrolateral parts.

(Figs 3 and 5). The topographic distribution of the a paramedian fissure exhibited the patchy activation activated patches in the granule cell layer was distinct pattern, whereas low and uniform Z-DC uptake at different rostra! caudai Ievels and in adjacent f&a. occurred in adjacent granule celf regions (Fig. 3H). For example, the granule eel1 layer on either side of The compfex patchy activation pattern in the

Table 2. Relative fluorodeoxyglucose uptake in flying-echolocating, echolocating-not-flying, and resting bats

Fiying- Echolocating-- Brain region echolocating not-flying Resting

Cingutate 2.26 f 0. i f I .73 i 0.08 I .46 i 0.02 cortex, ventral

Cingulate I .33 c 0.20 1.58 f0.13 I .23 + 0.04 cortex, dorsal

Auditory cortex 2.76-t-0.19 1.90+0.12 I. 77 + 0.07 Retrosplenial 2.40 c 0.14 I .46 + 0.08 1.36 + 0.05

cortex Medial 2.91 -+ 0.32 1.97 i-O.13 1.73 + 0.05

geniculate lnferior 3.73 f 0.48 2.85 c 0.36 2.86 + 0.09

colliculus Superior 2.96 + 0.27 2.13 -1-0.14 I .67 + 0.07

colliculus AIllbigLIUS 2.2f + 0.05 2.37+0.14 I.32 + 0.03

nucfnes

~aata are expressed as ratios of radioactivity in the brain regions relative to that in the corpus callosum. For the flying and echolo- eating-not-flying groups, data are means k S.E.M. for three bats in each condition, For the resting condition, n = 1 and the data are the mean + S.E.M. for three sections for each region. Repeated measures ANOVA indicates a significant difference between the ftying and echol~ating-not-flyillg groups (P i 0.01).

Z-DG uptake in flying bats 1057

cerebellum was remarkably similar for all flying bats. Although the molecular layer of the cerebellum did not exhibit the intense patchy activity pattern ob- served in specific regions of the granule cell layer, certain regions of the molecular layer also displayed a non-uniform pattern of 2-DG uptake (Fig. 5).

Comparison of activity patterns in echolocating-not- flying and flying-echolocuting bats

Bats that were placed in a wire cage after injection of FDG emitted a similar number of biosonar pulses (mean _+ SEM 693 I f 1226 pulses/20 min) compared

Fig. 3(A-H). caption ouerleaf

10.58 C. E. DUNCAN and 0. W. HENSON

Fig. X(1 N)

Fig. 3. Autoradiograms of coronai sections through the brainstem of resting (Ieft panels) and flying (right panels) bats. Sections are progressively more caudal from top to bottom. Note the increased 24X uptake in the pontine reticular formation and nucleus ambiguus (AM) in the flying condition. Under resting conditions, note the uniformly low 2-DC uptake in the forebrain but the relatively high uptake in certain

brainstem structures.

to flying bats (8972 5 1273 pulses/20 minf. The same forebrain and brainstem regions that showed markedly increased 2-DG uptake in response to flight were also heavily labeled in flying bats after injection of FDG (Figs 6-S). By contrast, forebrain regions that showed high FDG uptake in flying bats exhibited significantly less uptake in the bats that emitted thousands of biosonar pulses whiie hanging in the wire cage (Figs 6 and 7; Table 2). For example, the relative uptake of FDG in the auditory cortex, cingu- late cortex and medial geniculate body was signifi- cantly less in the echolocating-not flying compared to the flying bats (Table 2). In one of the echolocat- ing-not-flying bats, the relative FDG uptake in corti-

cal regions was atmost indistinguishable from the resting control (Fig. 8). The FDG uptake patterns in the brainstem were similar in the flying compared with the emitting-not-flying bats (Figs 7 and 8). For example, the inferior colliculus and nucleus ambiguus were densely labeled for both conditions.

DISCUSSION

The neurobiological basis of behavior involves the expression of specific spatial and temporal patterns of neural activity. Assessment of 2-DG uptake by auto- radiographic analysis provides a measure of spatial

2-DG uptake in flying bats 1059

activation patterns in specific experimental settings. assessment of 2-DG uptake can provide a useful This approach has proven especially useful for study- index of regional brain activity in freely behaving ing repetitive sensory stimulation and motor acti- animals.’ Interpretation of 2-DG uptake data in vation.3.6.9.‘0.“,2’,46,50 A valuable aspect of functional terms of electrical activity of neurons is not possible, mapping with 2-DG uptake is that global assessment because glial cells contribute to the accumulation of of brain activity patterns is possible. Although 2-DG the compound in neural tissue.‘**‘(’ In addition, in- uptake is not a universal functional indicator,‘*gi6 terpretation of 2-DG uptake data is complicated by

Fig. 4. Details of autoradiograms through the inferior colliculus of resting (left panels) and flying (right panels) bats. Sections are progressively more caudal from top to bottom. Photomicrographs were

under-exposed to illustrate the patterns of 2-DG uptake.

1060 Ci. E. DUNCAN and 0. W. HENSON

Fig. 5. Patch-like activation patterns within the granule cell layer of the cerebellum of a flying bat. (A) Detail of autoradiogram through the vermis. (B) Methyl Green-pyronin-stained section adjacent to the

section that produced the autoradiogram in (A).

the fact that both excitatory and inhibitory processes in neurons require energy.’ Despite the limitations of functional mapping with 2-DG, this approach can provide information on brain activity patterns in freely behaving animals that would be difficult or impossible to obtain with other methods.

take under resting conditions were the inferior col- liculus. superior olivary complex, cochlear nucleus. pontine central gray, and vestibular nuclei. These same regions also exhibit high 2-DG uptake in the rat brain under resting conditions.“,5’

In the present work, we found that 2-DG uptake in the forebrain of resting mustached bats was rela- tively low and minimal regional variations were ob- served. This contrasts with relatively high 2-DC uptake and considerable regional variations in the forebrain of rats under basal conditions.9.“.‘5~s’ In the control condition, the bats were seemingly ‘alert’, as indicated by frequent movements of the head and some audible chirping. Thus, the uniformly low 2-DG uptake in forebrain structures in resting bats, com- pared to resting rats, cannot be accounted for simply by a low ‘state of alertness’.

Although forebrain structures displayed uniformly low 2-DG uptake under resting conditions, certain regions of the brainstem exhibited relatively high levels of uptake. Regions exhibiting high 2-DG up-

During flight, mustached bats continuously emit biosonar pulses at rapid rates. Each pulse contains a relatively long, c. 7-25 ms, constant frequency (CF) component that is preceded and terminated by fre- quency modulated (FM) components. In addition. each component is represented by a series of four harmonics, of which the second (CF = 60 kHz) and the third (CF = 90 kHz) are the most intense. All four harmonics, however, appear to be important since many neurons are combination sensitive, i.e. exci- tation is dependent on the presence of more than one harmonic.i7~29~38~39~s3~5g~59 Recordings of brain potentials or cochlear microphonic potentials have shown that the ear is almost continuously stimulated when the animals are actively echolocating. Under flight con- ditions of the present study, bats emitted 7000-9000 biosonar pulses during a 20-min flight period and it

Fig. 6. FDG uptake patterns in coronal sections of the forebrain of emitting-not-flying and flying bats. Note relatively greater uptake in the ventral cingulate cortex, auditory cortex, medial geniculate nucleus. and superior colliculus in the flying bat. The number of pulses emitted (in a 20-min period) by the

emittinggnot-flying bat and flying bat were, respectively. 7120 and 8100.

2-DG uptake in flying bats 106I

Fig. 6(A-H). Continued overleaf

6(1 P)

2-DG uptake in flying bats 1063

Fig. 7(A-H). Caption on page 1065

1064 G. E. DUNCAN and 0. U’. HENSON

Fig. 7(1-P). Caption opposite

2-DG uptake in flying bats

Flying Emitting not flying

Resting

Fig. S--continued ouerleaf

_“.. ~.- ~11

Fig. 7. FDG uptake patterns in coronal sections of the brainstem of emitting--not-flying and flying bats. Note the similar patterns of FDG uptake in most structures for the two conditions but in the cerebellum,

activity patterns are distinctly different.

Flying Emitting

FC

CB

Resting

Fig. (i-c~orrtinud

1066

Z-DG uptake in flying bats 1067

is certain that many echoes were produced by each pulse. It is interesting to note that flying mustached bats continuously adjust their sonar emissions to compensate for pulse-echo overlap,“j Doppler shifts,47+4s signal intensity levels,30 and apparently changing properties of the inner ear.25~26~27~28 Thus, the dramatic alterations in brain activity patterns indi- cated from the present study is consistent with the high levels of sensory stimulation and information processing necessary for flight and echolocation.

In flying bats, all regions of the classical ascending auditory system showed marked activation. In the inferior colliculus, a distinctive pattern of 2-DG uptake was found. The activation patterns of the inferior colliculus of flying bats were different from those associated with presentation of the second harmonic of the FM component (FM2) and sequen- tial presentation of the FM2 and FM 1 harmonics in the 2-DG study of O’Neill et ~1.~’ In that study, the dorsal cortex and anterolateral division of the inferior colliculus were preferentially labeled and the medial division of the structure was not. By contrast, in our flying bats relatively Iow 2-DG uptake was found in the dorsal cortex and the medial division of the inferior colliculus was densely labeled. The differ- ences observed in the topographic activation patterns of 2-DG uptake between the present study and those described by O’Neill et ~1.~~ are probably due to the much greater complexity of the natural acoustic signals compared with the artificial ones.

Consistent with marked activation of the auditory system, the medial geniculate body showed high 2-DG uptake. Although high 2-DG uptake was found in the entire medial geniculate, the dorsal and medial divisions were more densely labeled than the ventral and lateral parts of the nucleus (Fig. 2). This activation pattern is congruent with electrophysio- logical data that demonstrated preferential dorsal and medial localization of neurons of the medial geniculate that have special properties important for target ranging and extraction of velocity information from pulse-echo perception.38.39 The specific pattern of 2-DG uptake observed in the medial geniculate of flying bats is also consistent with the topography of inputs from the inferior colliculus described for the mustached bat.‘*

In the 2-DG study of O’Neill et aL3’ presentation of artificial acoustic stimulation sufficient to increase 2-DG uptake in the inferior colliculus was not asso& ated with increased uptake in the auditory cortex or medial geniculate body of the mustached bat. The marked activation of 2-DG uptake in widespread regions of the auditory cortex and medial geniculate

of the flying bat, compared to the apparent lack of change in these regions in the work of O’Neill et aL3’ may be related to differences between active and passive listening. In this regard, our data (Table 2; Figs 6 and 8) show that bats hanging in a wire cage (unrestrained) and emitting biosonar pulses compar- able in number to flying bats exhibited significantly lower 2-DG uptake in the auditory cortex and medial geniculate body compared to flying bats.

Differences in brain activity patterns between flying bats and pulsing-caged bats may be accounted for by a number of factors. Although the echolocating-not flying bats emitted comparable numbers of pulses as flying bats, it must be assumed that the acoustic exposure during flight was much richer than in the cage. The flying bats undoubtedly received a complex array of echoes that were Doppler-shifted to varying degrees; any change in flight speed would produce changes in the amount of Doppler-shift, and echoes from off-center targets would be shifted to a different degree than those from targets directly ahead. In addition, the flying bats must have received a broad spectrum of different echo-delays as the primary targets (walls, ceiling, floor) were imaged. Although the caged bats heard echoes from objects outside of the cage (since they readily responded when objects were waved) the echo delays for the emitting-not flying bats would be over a more restricted distance band than in flight. In addition, since mustached bats are known to echo-intensity compensate,3’ it is to be expected that the pulses emitted by the caged animals were less intense than for the flying bats. It also seems likely that flying bats were more attentive to the echoes than the emitting-not-flying bats and that sensory-motor integration required for flight contrib- utes to the differences observed between these con- ditions.

In addition to the auditory cortex, other cortical regions accumulated high levels of 2-DG in flying bats. These included certain parts of the cingulate, frontal and retrosplenial cortices. The cingulate cor- tex has been implicated in the initiation of biosonar emissions in the mustached bat.” In the present study, high 2-DG and FDG uptake was found in the ventral part of the cingulate cortex in flying bats, However, in bats emitting thousands of biosonar pulses but not flying, the cinguiate cortex was not heavily labeled. These data suggest that activation of the cingulate cortex in flying bats is not simply a function of vocalization.

The amygdala is well developed in bats and has been shown to receive auditory projections.32x33l60 This structure exhibited low 2-DG uptake in flying bats

Fig. 8. FDG uptake patterns in horizontal sections of resting, emitting-not-flying, and flying bats. Note the similar labeling patterns for the resting and emitting-not-flying bats and the much greater relative FDG uptake in select regions of the flying bat. The number of pulses emitted by the resting, emitting-not-flying and flying bats were, respectively, 313,938l and 9850 pulses in a 20-min period. Note that the patterns of FDG uptake in cortical regions of the resting and emitting-not-flying bats are similar,

but there is markedly greater FDG uptake in select regions of the Rying bat.

1068 Ci. E. DUNCAN and 0. W. HENSON

and thus its role in flight control or echolocation is

questionable. A number of brainstem regions have been impli-

cated in the emission of biosonar pulses of bats; these include parts of the central gray.” reticular for- mation, lateral lemniscus,49 and nucleus ambiguus.4’ These areas all showed high metabolic activity in flying animals and in the emitting-not-flying bats, It is usually difficult to relate specific regions to a given sensory or motor function but the large nucleus ambiguus in the mustached bat (Fig. 3) is known to contain the motor neurons for the muscles of the larynx.‘” The dramatic increase in the metabolic activity in this nucleus is consistent with laryngeal

muscle activity associated with emission of many thousands of pulses in our experiments. Vocalization in rats has also been shown to be associated with increased 2-DG uptake in the cenlral gray and nucleus ambiguus.”

The superior colliculus is usually associated with visual perception. In mustached bats the superior colliculus is well developed even though these animals use biosonar primarily for envjronnlental imaging. Neuroanatomical studies have shown that the su- perior colliculus of the mustached bat receives signifi- cant inputs from the inferior colliculus and nucleus of the lateral lemniscus.s~7~‘K Electrophysiological studies have also implicated the superior colliculus of the horseshoe bat in echolocation and acoustic-guided responses,?‘.4’.““.57 These published data correspond with the marked increase in metabolic activity of the superior colliculus during flight, as indicated by the present 2-DG study. The uptake of FDG in the superior colliculus was significantly less in the emit- ting-not-flying bats compared with the flying bats.

Autoradiograms of 2-DG uptake in the cerebellum of flying bats revealed a complex activation pattern. In highly selective regions of the granule cell layer, intense patchy patterns of Z-DG uptake were ob- served. Similar patterns of 2-DG uptake have been found in the rat cerebellum after electrical stimulation of the motor cortex.” To our knowledge, the present 2-DG study is the first description of global patterns of functional activity in the cerebellum of a freely behaving animal. The functional organization re- vealed is consistent with electrophysiological studies that have shown that the mammalian cerebellum contains a ‘fractured somatotopy’, where different musculature is represented in discontinuous patches in the granule cell layer.”

CONCLUSIONS

The present investigation provides an overview of regional 2-DG uptake in the brains of flying bats. Determination of the specific role of certain brain regions in acoustico-motor integration that were acti- vated during flight, such as the hippocampus, reticu- lar formation, frontal and cingulate cortices, will require further study. The results provide a foun- dation for further detailed analysis of the functional role and organization of specific brain regions in the mustached bat.

Acknowledgements-This work was supported by NIX grants DC 00918 and DC 0114 from the National Institute on Deafness and Other Communicative Disorders. We thank Dr Miriam Henson for critical reading of the manuscript and Art Keating and En-Qi Jiang for technical assistance.

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(Accepted I Orrohei 19931